The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide: pidotimod(PTD)

Abstract

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-α. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs.

Figure 2. The curve of the DCs’ proliferation after treatment with a range of concentrations of PTD for 48h determined by method of MTS.

Figure 3. Morphology of the BMDCs before and after treatment with PTD under a light microscope (A) ( × 400). The images of DC2.4 cells before and after treatment with PTD with SEM (B) ( × 3500).

Figure 4. TEM for changes of lysosomes inside the DCs2.4 cells before and after treatment with PTD.

Figure 5. The ACP activity of the DCs2.4 cells after treatment with 800 ug/ml PTD for 48 h.

Figure 6. Confirmation by FCM of the DCs2.4 cells’ phagocytic effect. Results represent the mean ± SD of three independent samples.

Figure 7. Immunohistochemical staining of DCs2.4 cells after treatment with PTD.

Figure 8. Upregulation of key surface molecules on BMDCs (A) and DCs2.4 cells (B) after treatment with PTD for 48 h. The cells were respectively collected and stained with mAbs to CD40, CD86, and MHCII. Expression of surface markers was analyzed by FCM, which was displayed respectively by the single parameter diagrams. The values shown in the proðles were the gated % and the mean ñuorescence intensity indexes (MFI). Results represent the mean ± SD of three independent samples.

Figure 9. (A, B) The production of IL-12p70, IL-12p40 by the DCs 2.4 cells after treatment with PTD. The histograms above showed the IL-12p70, IL-12p40 production levels after PTD stimulation. Results represent the mean ± SD of three independent samples. (C) The production of TNF-α by the DCs 2.4 cells after treatment with PTD. Results represent the mean ± SD of three independent samples.

Figure 1. The CD11c+ cell puriðcation with MACS. After co-culture for 6 d, the purity of CD11c+ cells were examined and the percentage were over 80%. Followed by purification with MACS, the CD11c+ cells were enriched and the results of FCM showed that the purity CD11c+ cells approached 95%.